Selective inactivation of two components of the multiprotein transcription factor TFIIIB in cycloheximide growth-arrested yeast cells.

Following protein synthesis inhibition in cycloheximide growth-arrested yeast cells, the rates of tRNA and 5 S RNA synthesis decrease with apparent half-times of about 20 and 10 min, respectively. This effect is mimicked by extracts of treated cells, and the impairment of tRNA gene transcription activity that is observed in vitro parallels the in vivo inactivation of RNA polymerase III transcription. As revealed by experiments in which partially purified class III transcription factors were singly added to extracts of treated cells, only the activity of the multiprotein transcription factor TFIIIB is severely impaired after 3 h of cycloheximide treatment. Similar assays carried out in an in vitro transcription system in which TFIIIB activity was reconstituted by a combination of the TATA box-binding protein (TBP), the 70-kDa component TFIIIB70, plus a partially purified fraction known as B" have shown that the latter two components are both necessary and sufficient to restore control levels of transcription. Their activity, but not TBP activity, is considerably reduced in extracts of treated cells. TFIIIB70 and a component of fraction B" thus appear to be the selective targets of the down-regulation of polymerase III transcription that is brought about by cycloheximide. A substantial depletion of the TFIIIB70 polypeptide was detected by Western immunoblot analysis of extracts derived from cycloheximide growth-arrested cells, indicating that the inactivation of this TFIIIB component results primarily from its enhanced destabilization under conditions of protein synthesis inhibition.

A novel RNA polymerase III transcription factor fraction that is not required for template commitment.

We have identified and partially characterized a novel class III transcription factor fraction (TFIIIE) from yeast nuclear extracts. TFIIIE is functionally distinct from the standard yeast transcription factor fractions, TFIIIB and TFIIIC. It is also different from either of the TFIIIB subfractions, B' and B". TFIIIE is essential for specific transcription of both tRNA and 5 S RNA genes, its activity is sensitive to proteinase K, and it exhibits an apparent sedimentation coefficient of 4.0 S when analyzed on glycerol gradients. In the case of a tRNA gene, TFIIIE does not play a role in the formation of stable preinitiation complexes containing TFIIIB and TFIIIC. It is required for single as well as multiple rounds of transcription, however. Thus, TFIIIE is involved in the utilization of stable transcription complexes, but its action is not restricted to reinitiation events.